Rna molecules and uses thereof

Abstract

The invention relates to a method of designing a short RNA molecule to increase the expression of a target gene in a cell through the down-regulation of a non-coding RNA transcript, said method comprising the steps of: a) obtaining the nucleotide sequence of the coding strand of the target gene, at least between 200 nucleotides upstream of the gene's transcription start site and 200 nucleotides downstream of the gene's transcription start site; b) determining the reverse complementary RNA sequence to the nucleotide sequence determined in step a); and c) designing a short RNA molecule which is the reverse complement or has at least 80% sequence identity with the reverse complement of a region of the sequence determined in step b); wherein said method does not include a step in which the existence of said non-coding RNA transcript is determined; as well as to such short RNA molecules and uses thereof.

1 . A method of designing a short RNA molecule to increase the expression of a target gene in a cell through the down-regulation of a non-coding RNA transcript, said method comprising the steps of:
 a) obtaining the nucleotide sequence of the coding strand of the target gene, at least between 200 nucleotides upstream of the gene's transcription start site and 200 nucleotides downstream of the gene's transcription start site;   b) determining the reverse complementary RNA sequence to the nucleotide sequence determined in step a); and   c) designing a short RNA molecule which is the reverse complement or has at least 80% sequence identity with the reverse complement of a region of the sequence determined in step b); wherein said method does not include a step in which the existence of said non-coding RNA transcript is determined.   
     
     
         2 . The method of  claim 1 , wherein the target gene is a pluripotency-inducing gene. 
     
     
         3 . The method of  claim 1 , wherein the region defined in c) includes the reverse complement of the gene's transcription start site. 
     
     
         4 . The method of  claim 1 , wherein the short RNA molecule is from 16 nucleotides to 30 nucleotides in length. 
     
     
         5 . The method of  claim 1 , which further comprises the step of generating a double-stranded siRNA molecule which incorporates said short RNA molecule. 
     
     
         6 . The method of  claim 5 , wherein each strand of said double-stranded siRNA molecule is 16 to 30 nucleotides in length and wherein said molecule is hybridised over a length of at least 12 nucleotides. 
     
     
         7 . The method of  claim 1 , wherein the short RNA molecule is 21 nucleotides in length. 
     
     
         8 . The method of  claim 1 , wherein the short RNA molecule is the reverse complement or has at least 95% sequence identity with the reverse complement of a region of the sequence determined in step b). 
     
     
         9 . The method of  claim 1 , wherein step a) comprises obtaining the nucleotide sequence of the coding strand of the target gene, at least between 500 nucleotides upstream of the gene's transcription start site and 500 nucleotides downstream of the gene's transcription start site. 
     
     
         10 . A method of increasing the expression of a target gene in a cell through the down-regulation of a non-coding RNA transcript, said method comprising the steps of:
 a) obtaining the nucleotide sequence of the coding strand of the target gene, at least between 200 nucleotides upstream of the gene's transcription start site and 200 nucleotides downstream of the gene's transcription start site;   b) determining the reverse complementary RNA sequence to the nucleotide sequence determined in step a);   c) designing a short RNA molecule which is the reverse complement or has at least 80% sequence identity with the reverse complement of a region of the sequence determined in step b); and   d) contacting the cell with said short RNA molecule, wherein said method does not include a step in which the existence of said non-coding RNA transcript is determined.   
     
     
         11 . One or more computer-readable media comprising computer-executable instructions to instruct a computing system to:
 a) receive a nucleotide sequence of a coding strand of a target gene, at least between 200 nucleotides upstream of the gene's transcription start site and 200 nucleotides downstream of the gene's transcription start site;   b) determine a reverse complementary RNA sequence to the nucleotide sequence received in step a); and   c) output information to construct a short RNA molecule designed to increase expression of the target gene in a cell through the down-regulation of a non-coding RNA transcript wherein the short RNA molecule is the reverse complement or has at least 80% sequence identity with the reverse complement of a region of the sequence determined in step b); wherein the instructions do not comprise instructions to call for determining existence of the non-coding RNA transcript.   
     
     
         12 . The one or more computer-readable media comprising computer-executable instructions to instruct a computing system of  claim 11 , further comprising instructions to output the information to construct a short RNA molecule onto a computer-readable medium. 
     
     
         13 . A method of maintaining or increasing the differentiation potential of a population of cells by up-regulating a target gene in said cells, wherein said target gene is a pluripotency-inducing gene and wherein said method comprises contacting said cells with a short RNA molecule which specifically down-regulates a target RNA transcript in said cells, wherein said target RNA transcript:
 i) is transcribed from
 a) either strand of a locus up to 100 kb upstream of the target gene's transcription start site, 
 b) either strand of a locus up to 100 kb downstream of the target gene's transcription stop site; or 
 c) either strand of a locus which interacts physically with the target gene; and 
   ii) comprises a sequence which is antisense to a genomic sequence located between 100 kb upstream of the target gene's transcription start site and 100 kb downstream of the target gene's transcription stop site.   
     
     
         14 . The method of  claim 13  wherein the target RNA transcript comprises a sequence which is antisense to a genomic sequence which comprises the target gene's transcription start site. 
     
     
         15 . The method of  claim 13 , wherein the target gene is selected from the group consisting of KLF4, POU5F1, SOX2, MYC, NANOG and LIN28. 
     
     
         16 . The method of  claim 15  wherein the target gene is KLF4. 
     
     
         17 . A method of producing an RNA molecule which comprises performing a method as claimed in  claim 1  and synthesising the RNA molecule designed thereby. 
     
     
         18 . A short RNA molecule having any one of the sequences set out in Tables 1 to 3 herein. 
     
     
         19 . The method of  claim 10 , wherein the target gene is selected from the group consisting of KLF4, POU5F1, SOX2, MYC, NANOG and LIN28.